MicroRNAs (miRNAs), typically 18 to 25 nt in length, are non-protein-coding RNAs that can inhibit the translation of target mRNAs (Croce and Calin. 2005. miRNAs, cancer, and stem cell division. Cell 122(1): 6-7). miRNAs directly or indirectly regulate a wide range of genes, and are involved in a remarkable spectrum of biological pathways including cell development, proliferation and apoptosis (He and Hannon. 2004. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5(7): 522-31, Alvarez-Garcia and Miska. 2005. MicroRNA functions in animal development and human disease. Development 132(21): 4653-62). As of September 2009, 10883 miRNA entries from vertebrates, flies, worms, plants, and viruses, including 721 human miRNAs and 579 mouse miRNAs, have been annotated (miRBase, Release 14) in the Sanger Institute miRNA sequence database (Griffiths-Jones, Saini, van Dongen and Enright. 2008. miRBase: tools for microRNA genomics. Nucleic Acids Res 36(Database issue): D154-8); the function of many miRNAs is unknown.
Materials and Methods that can detect and quantify miRNAs with high sensitivity and specificity are useful. Cellular miRNA profiles can offer insights into gene expression, and allow the determination of the species, tissue types, and developmental stages of tissue samples. Further, the detection and quantification of miRNAs can lead to the discovery of novel, miRNA-based diagnostic/prognostic biomarkers and therapeutic agents.
However, detection of mature miRNAs is difficult due to several reasons. First, miRNAs are difficult to detect because they are relatively short nucleic acid molecules (on average, only about 22 bases in length). In addition, these short sequences can be present in sequences other than mature miRNA, such as pre-miRNA, pri-miRNA, genomic DNA and mRNA. Further, it is difficult to distinguish miRNAs within the same family, as these miRNAs usually differ from each other only in terms of one or a few nucleotides. Moreover, the melting temperatures (Tm) of miRNAs can vary greatly, from about 55° C. to 90° C.
At present, although a wide spectrum of miRNA detection techniques have been developed, there is a lack of high-throughput profiling assays that can sensitively and specifically detect miRNAs. Conventional techniques for miRNA profiling include Northern hybridization, cloning, and microarray analysis. (Wang, Ach and Curry. 2007. Direct and sensitive miRNA profiling from low-input total RNA. RNA 13(1): 151-9, Wang and Cheng. 2008. A simple method for profiling miRNA expression. Methods Mol Biol 414: 183-90, Shingara, Keiger, Shelton, Laosinchai-Wolf, Powers, Conrad, Brown and Labourier. 2005. An optimized isolation and labeling platform for accurate microRNA expression profiling. RNA 11(9): 1461-70, Nelson, Baldwin, Scearce, Oberholtzer, Tobias and Mourelatos. 2004. Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1(2): 155-61). These techniques are not as sensitive or specific, when compared to quantitative real-time reverse transcription PCR (qRT-PCR).
Several qRT-PCR-based methods have been developed for detecting and quantifying miRNAs (Li, Yao, Huang, Wang, Sun, Fan, Chang, Li, Wang and Xi. 2009. Real-time polymerase chain reaction microRNA detection based on enzymatic stem-loop probes ligation. Anal Chem 81(13): 5446-51, Varkonyi-Gasic, Wu, Wood, Walton and Hellens. 2007. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3: 12, Ro, Park, Jin, Sanders and Yan. 2006. A PCR-based method for detection and quantification of small RNAs. Biochem Biophys Res Commun 351(3): 756-63). The current reverse transcriptase quantitative polymerase chain reaction assays (RT-qPCR), which use SYBR Green, are lacking in specificity and sensitivity, as SYBR Green detects all forms of nucleic acids, including double-stranded DNA, double-stranded RNA, single-stranded RNA and DNA, although the detection sensitivity of double-stranded RNA, single-stranded RNA and DNA is lower than that of double-stranded DNA.
The most frequently used qRT-PCR-based method, developed by Chen et al. (Chen, Ridzon, Broomer, Zhou, Lee, Nguyen, Barbisin, Xu, Mahuvakar, Andersen, Lao, Livak and Guegler. 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20): e179), includes two main steps: reverse transcription of miRNAs using stem-loop RT primers, followed by a TaqMan® PCR analysis.
However, the Chen et al. method has several limitations. According to Chen et al., profiling each target miRNA requires a target-specific TaqMan® probe and a target-specific RT primer. As a result, the cost of making hundreds of target-specific probes and RT primers during miRNA screening tests can be prohibitive. In addition, the Chen et al. method is procedurally complex. Profiling each miRNA requires an RT reaction; otherwise, if only one RT reaction is performed, all miRNA-specific RT primers need to be mixed together. Further, hundreds of target-miRNA specific TaqMan® probes need to be added separately in order to detect or quantify miRNA. Moreover, RT primers used in the Chen et al. method only have a 6-nt-sequence that base-pairs with the target miRNAs. As a result, the RT primers may hybridize to, and prime, other RNAs during the RT reaction (Tang, Hajkova, Barton, Lao and Surani. 2006. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res 34(2):e9). Accordingly, improved methods for profiling miRNAs are needed.